ARF GTPases are key regulators of vesicle transport steps and thereby control a broad range of processes in eukaryotic cells, including the maintenance of intracellular compartments and the exocytosis of proteins and neurotransmitters. Research in this proposal focuses on an integrated understanding of ARF function in the processes of vesicular coat assembly and regulated disassembly. Vesicle assembly is triggered by the GTP-dependent recruitment of vesicular coat complexes by ARF. We will explore the mechanism of GDP-to-GTP conversion on ARF1 by determining the crystal structure of a long-sought-after intermediate in the nucleotide exchange reaction: ARF1-GDP complexed with its Sec7 catalyst, trapped using the fungal toxin, Brefeldin: high-quality crystals (2.4 Angstroms resolution) are in hand. We will explore the process of coat recruitment through analyses of ARF-coat interactions, involving the biochemical dissection of heptameric (550 kDa) COPI and tetrameric (400 kDa) COPII coats. For COPI, we have established protein expression systems for six subcomplexes, and plan now to determine the structure of ARF1 bound to an "active" COPI subcomplex. For the COPII coat, we previously determined the crystal structure of the inner shell of the coat bound to the ARF homolog, Sar1. We have now dissected the COPII outer shell biochemically, and will determine the crystal structure of a core outer-shell complex: high-quality crystals (2.6 Angstroms resolution) are in hand. Finally, we will explore vesicular coat disassembly by focussing on the GTP hydrolysis reaction that triggers this process. We previously discovered that COPI accelerates GTP hydrolysis in the ARF1-ARFGAP complex by 300-fold or more. We will investigate the basis for this effect by biochemical and crystallographic analysis of COPI core bound to ARF1-ARFGAP in ground- and transition-state complexes. In the COPII system, maximal rate acceleration of the GTPase requires catalytic contributions from both the inner and outer shells of the coat. We recently defined an assembly capable of maximal rate acceleration that includes inner (Sec23) and outer (Sec31 fragment) shell molecules complexed with Sar1 GTPase. We have obtained small crystals of this complex that diffract to at least 2.8 Angstroms resolution in-house, which we are working to improve. These studies will provide insight to the processes of GTP-dependent assembly of vesicular coat complexes and coat-controlled GTP hydrolysis triggering disassembly.